Background chemical noise in a mass spectrum can be particularly problematic. The background chemical noise observed in mass spectra often has a periodic nature especially at mass to charge ratios less than 1000. As will be understood by those skilled in the art, all elements have near integral masses. Carbon-only graphite has, by definition, an exact integer mass of 12 and all other molecules of the same nominal mass will have an exact mass which is not quite an exact integer value but yet which is only slightly higher or lower than the corresponding mass of carbon-only graphite.
The most mass sufficient ions formed from organic and biological molecules are saturated hydrocarbons and the most mass deficient ions formed from organic and biological molecules are saturated bromocarbons. Saturated hydrocarbons have a mass sufficiency of about 0.1%. Accordingly, a saturated hydrocarbon with a nominal mass of 100 will have an exact mass of about 100.1 and likewise a saturated hydrocarbon with a nominal mass of 200 will have an exact mass of about 200.2. Saturated bromocarbons have a mass deficiency of about 0.1%. Accordingly, a saturated bromocarbon with a nominal mass of 100 will have an exact mass of about 99.9 and likewise a saturated bromocarbon with a nominal mass of 200 will have an exact mass of about 199.8. As a result, at a nominal mass of 200 singly charged ions can be expected to have exact masses which fall within a relatively narrow mass to charge ratio range of 199.8 to 200.2. Similarly, at a nominal mass of 201 singly charged ions can be expected to have exact masses which fall within a similar relatively narrow mass to charge ratio range 200.8 to 201.2. It will therefore be appreciated that no singly charged ions having exact masses in the range 200.2 to 200.8 will be observed. Accordingly, at relatively low mass to charge ratios the chemical background noise in mass spectra (which is predominantly singly charged) typically exhibits a distinct periodicity of approximately 1 atomic mass units (amu).
For singly charged ions having mass to charge ratios of 500 or more, the range of forbidden exact masses theoretically shrinks to zero and hence it might be expected that the chemical background noise would no longer exhibit a periodicity of approximately 1 atomic mass unit. However, in practice, saturated hydrocarbons and saturated bromocarbons are rarely encountered when mass analysing biochemical samples such as proteins and peptides. Accordingly, the chemical background noise in mass spectra relating to biochemicals or biomolecules commonly exhibits a distinct periodicity of approximately 1 atomic mass unit at mass to charge ratios in excess of 500. Indeed, mass spectra commonly exhibit a distinct periodicity of approximately 1 atomic mass unit at mass to charge ratios up to about 2000 and periodic background noise may, in some circumstances, be observed at mass to charge ratios in excess of 2000.
Most non-halogenated organic molecules have a mass sufficiency in the range 0.0% to 0.1%. Therefore, assuming that halogenated compounds are absent, then it will be appreciated that the chemical background noise can still be expected to have a periodicity of approximately 1 atomic mass unit at mass to charge ratios up to 1000. Indeed, in practice, chemical background noise having a periodicity of approximately 1 atomic mass unit is commonly observed when mass analysing ions derived from biomolecules having mass to charge ratios up to about 2000.
Many mass spectrometric techniques have detection limits which are restricted or otherwise compromised by the presence of chemical background noise. The precise chemical nature of the background noise is often unknown and the presence of unwanted chemical background noise can adversely affect mass measurement accuracy especially if an analyte signal is not fully resolved due to chemical background noise.
Chemical background noise may, for example, arise from impurities in solvents, analytes or reagents. Impurities in drying or nebulizing gases can also cause chemical background noise. Contamination of the solvent or analyte delivery system or contamination within or on the surfaces of an ionisation chamber can be a further source of chemical background noise.
In Atmospheric Pressure Ionisation (“API”) ion sources such as Electrospray (“ESI”), Photo Ionisation (“APPI”) or Atmospheric Chemical Ionisation (“APCI”) ion sources, chemical background can arise from the clustering of solvent and analyte ions. In Chemical Ionisation (“CI”) ion sources chemical background can arise from self-adduction of reagent gas ions or from reagent gas contamination. In Matrix Assisted Laser Desorption Ionisation (“MALDI”) ion sources chemical background can arise from matrix cluster ions.
In general the chemical background noise observed in mass spectra tends to be complex in nature and may only be partially mass resolved. The chemical background noise tends to be singly charged and to have a periodic nature with a repeat unit of approximately 1 atomic mass unit. Amino acids have a mass sufficiency which varies from about 1.00009 to about 1.00074, with a mean mass sufficiency of approximately 1.00047. Accordingly, biological samples commonly exhibit a periodicity of approximately 1.0005 atomic mass units (Daltons).
A known approach to reducing the effects of periodic background chemical noise in a mass spectrum is to transform the mass spectrum into the frequency domain and then to filter out noise components. Signals in the transformed spectrum which are considered to represent noise can then be removed at certain calculated frequencies. An inverse transform is then applied to the transformed spectrum in order to reproduce a mass spectrum which exhibits reduced periodic background noise.
Non-sinusoidal periodic noise will appear as a series of sharp spikes and harmonics in the frequency domain or transformed spectrum. Ion signals however, since they are of relatively small extent in mass to charge ratio, will tend to be smeared out across a relatively broad range of frequencies. The different characteristics of signal and noise in the frequency domain or transformed spectrum can in theory at least be used to allow the contribution of chemical background noise in the overall spectrum to be reduced. However, one problem with frequency domain filtering is that the unprocessed time of flight mass spectra data will comprise intensity data which is equally spaced in time due to the acquisition electronics. Since flight time in a Time of Flight mass analyser is proportional to the square root of the mass to charge ratio of the ions, the intensity data will be unequally spaced with respect to mass to charge ratio. Accordingly, prior to filtering the data in the frequency domain or transformed spectrum, it is first necessary to process the mass spectral data such that the intensity data is more equally spaced with respect to mass to charge ratio. It is known to use an interpolation algorithm to process the intensity data so that the data becomes equally spaced with respect to mass to charge ratio. However, disadvantageously, the use of an interpolation algorithm significantly increases the overall processing time.
In addition to increasing the overall processing time, the known approach of reducing periodic noise in a mass spectrum by filtering the data in the frequency domain suffers from the problem that the application of a filter to the frequency domain data to remove noise components can actually result in additional noise and discontinuities being present into the mass spectrum after data in the frequency domain has been transformed back into the mass to charge ratio domain. As a result, artefacts or spurious peaks can appear in the final processed mass spectrum which were not present in the original mass spectral data.
Another problem with the known frequency domain filtering approach is that a proportion of the desired analyte signal will have frequency components which are similar or identical to the frequency components corresponding to unwanted background noise. Accordingly, the removal of such components in the frequency domain can lead to distortion both of the analyte ion peak shape and also of the intensity of the analyte signal in the final processed mass spectrum.
A yet further problem with the known frequency domain filtering approach is in responding to changes in the characteristic of the background noise as a function of mass to charge ratio. The observed background noise in a mass spectrum often takes on a different nature in different portions of the mass spectrum i.e. the background noise is often observed to vary as a function of mass to charge ratio. If therefore a filter needs to change shape as a function of mass to charge ratio in response to the changing nature of the background noise, then the mass spectrum must first be divided up into a number of separate sections, each of which must then be treated or filtered slightly differently. However, discontinuities can then arise when a composite mass spectrum is subsequently reconstructed from the separate sections of data.
It is apparent therefore that the known frequency domain filtering approach suffers from a number of problems.
It is therefore desired to provide an improved method of reducing the effects of background chemical noise in mass spectra and in particular to reduce the effects of background chemical noise having a periodic nature.